Alcohol use disorders (AUDs) affect millions of Americans every year, at great personal, social, and economic cost. Women are particularly vulnerable to the negative health consequences of alcohol abuse, suffering neurological, cardiovascular, and liver damage at lower levels of alcohol abuse than men. Alarmingly, recent decades have seen a dramatic increase in the prevalence AUDs among the female population. Current treatment strategies are based on studies conducted predominantly in male animals, and little is known about sex differences in the brain's response to ethanol. A growing body of evidence suggests that estrogen receptors (ERs) are able to increase both the consumption of alcohol and the subjective pleasure (or reward) of the alcohol experience in female animals. In preliminary studies, our lab has demonstrated increased ethanol reward and binge-like drinking behavior in female mice treated with the ovarian steroid hormone estradiol. The amygdala, a brain region well established as a mediator of AUD development in animal models of ethanol addiction, expresses a high density of ERs. Furthermore, ERs regulate the expression of multiple genes that are known modulate the effects of alcohol on amygdalar neurotransmission. The objectives of this project are therefore: 1) Determine which of the classical ERs, ER? or ER?, is primarily responsible for estrogenic enhancement of ethanol reward and binge-like drinking behavior, 2) Investigate the effects of estrogen and ethanol on gene expression in the amygdala, and 3) Identify regions of the amygdala where ERs act to enhance ethanol-related behaviors in female mice. First, systemic treatment with the ER?- and ER?-specific agonists PPT and DPN will be used to determine which ER mediates estradiol's enhancement of ethanol reward and binge-like drinking behavior in the conditioned place preference (CPP) and drinking in the dark (DID) paradigms, respectively. Second, amygdalar tissue will be isolated from female mice treated with ER agonists and subjected to binge ethanol exposure in the DID paradigm, and real-time polymerase chain reaction (RT-PCR) analysis will measure the expression of key genes in subregions known to be involved in behavioral responses to ethanol. Third, RNAi knockdown of ER? or ER? expression in the central and medial amygdalar nuclei (CeA and MeA) will be used to localize ER effects on ethanol-related behaviors in naturally cycling female mice. The proposed project implements well-established behavioral and molecular techniques to investigate the role of amygdalar ERs in regulating the female brain's response to ethanol. This Research Training Plan will therefore make an important contribution to scientific understanding of AUD development in women while preparing the applicant for a successful academic career in ethanol research.